Immunoassay methods, in general, are based on the competition between a specific analyte, the amount of which is to be determined in a sample and a known amount of tracer, which is generally the analyte or appropriate analog thereof in labeled form, with the analyte and tracer competing for a limited number of available binding sites on a binder which is specific for the analyte and tracer.
If the concentration of tracer and binder is fixed and the only variable is the level of analyte, it is possible to establish an assay system for measuring the unknown level of analyte by determining the amount of bound and/or free tracer in the system. The values determined in the assay are compared with the values given by a range of known amounts of the analyte treated in the same manner, and by such comparison, it is possible to determine the amount of analyte in the sample.
In one such procedure, the binder is supported on a solid support, whereby the bound and free components of the assay, after incubation, may be easily separated by separation of the sample and the solid support.
In many cases, the tracers used in such assays require either instrumentation and/or treatment of the tracer in order to determine the tracer in the bound and/or free portion of the assay as a measure of analyte. Thus, for example, in an assay in which an enzyme is used as the label or marker for the tracer, the enzyme must be developed with a suitable developer. When a label or marker is a fluorescent material, the tracer in the bound and/or free portion is determined by the use of appropriate instrumentation for determining fluorescence.
An early test strip device is described by Deutsch, et al., in U.S. Pat. No. 4,361,537. In general, the device comprises a material capable of transporting a solution by capillary action, i.e., a wicking or chromatographic action. Different areas or zones in the test strip contain the assay reagents needed to produce a detectable signal as the analyte is transported to or through such zones. The device is suitable for chemical assays and binding assays and uses a developer solution to transport analyte along the strip.
Many alternatives to or variations on the Deutsch, et al. device have been disclosed. For example, Grubb, et al. (U.S. Pat. No. 4,168,146) describes the use of a porous test strip material to which is covalently bound an antigen-specific antibody. In performing the assay, the test strip is immersed in a solution suspected of containing an antigen, and capillary migration of the solution through the test strip is allowed to occur. As the antigen moves through the test strip it binds to the immobilized antigen-specific antibody. The presence of antigen is then determined by wetting the test strip with a second antigen-specific antibody to which a fluorescent or enzyme label is covalently bound. Quantitative testing can be achieved by measuring the length of the strip that contains bound and labeled antigen.
Tom, et al. (U.S. Pat. No. 4,366,241) describe a device comprised of a test zone (immunosorbing zone) and a liquid absorbing zone in liquid receiving relationship with the test zone. The immunosorbing or test zone has a member of an immunological pair (a "MIP") affixed to a solid support, where the MIP is inhibited from diffusing from the zone. In use, the analyte in the sample solution passes through the immunosorbing zone and binds to the MIP and is concentrated in the immunosorbing zone. The device includes a signal producing system for production of a detectable signal in the test zone in relation to the amount of analyte present in a sample. Tom, et al. teaches the use of the signal producing signal that provides for a detectable signal in the immunosorbing zone which can be compared to a signal level based on a standard having an unknown amount of analyte.
Weng, et al. (U.S. Pat. No. 4,740,468) describe another device and method for performing a specific binding assay. The assay involves both an immobile second binding member which binds to a mobile first binding member and an immobilized analog of the analyte which removes the unbound first binding member from the assay system prior to it being contacted to the detection site.
Greenquist, et al. (U.S. Pat. No. 4,806,311) describe a similar device wherein a first immobilized reagent, such as an analyte-analog is present in a reagent zone to remove free monovalent labeled-binding members from the assay system prior to the test samples contacting the detection layer reagents.
Ullman, et al. (EP 0 271 204 A2) describe a method of determining the presence of an analyte in a sample which is suspected of containing the analyte consisting of contacting a piece of bibulous material with a test solution containing the sample and a first specific binding pair member analogous to the analog. The material contains a second specific binding pair member capable of binding analyte to the first specific binding pair member. The second specific binding pair member is non-diffusively bound to the bibulous material at least at a portion thereof between the contact portion and a small situs on the piece separated from the contact portion. The surface area of the situs is substantially less than the material. The situs is capable of binding the first specific binding pair member not bound to the second specific binding pair member. The test solution is allowed to traverse the material by means of capillary migration and contact the situs. The situs is then examined for the presence of a specific binding pair member which is usually indicated by the presence of a detectable signal. The patent discloses that the signal can be directly detectable or the situs can be exposed to a signal producing means capable of interacting with the first specific binding pair member to produce a detectable signal at the situs in relation to the amount of analyte in the sample. May, et al. (E.P. 0 291 194 A1) disclose an analytical test device comprised of a hollow casing containing a dry porous carrier which communicates with the exterior of the casing such that a sample can be applied to the carrier. The device also contains a labeled specific binding reagent for an analyte which is freely mobile within the carrier. The device further contains an unlabeled specific binding reagent for the same analyte which is permanently immobilized in a detection zone on the carrier. The labeled reagent and detection zone are positioned such that when a liquid sample is applied to the device it can pick up the labeled reagent and thereafter permeate into the detection zone. The device also incorporates a means for indicating the extent, if any, to which the labeled reagent becomes bound in the detection zone. The binding of the labeled reagent can be observed by the user. The patent discloses that a plurality of detection zones arranged in a series on the porous solid phase material, through which the aqueous liquid sample can pass progressively, can also be used to provide a quantitative measurement of the analyte.
Ching, et al. (EP 299 428 A2) describe assay methods and devices utilizing colloidal particle labeled binding materials which are chromatographically mobile and capable of producing visual detectable signals. The method includes contacting the sample with a chromatographic medium having at least one reaction site which includes an immobilized reagent capable of binding a colloidal particle labeled material (which is capable of producing a detectable response) and the substance in the sample. The colloidal particle labeled material is transported on the chromatographic medium such that a portion of the material is transported to the reaction site and is bound thereto. The method that is taught in the patent is concluded by determining the detectable response produced by the colloidal material at the reaction site which indicates the presence or amount of the substance in the sample. An embodiment (competition-type) is described wherein the quantity of analyte present in the sample determines the amount of labeled first reagent which binds at the first zone. The patent teaches that adjustments to the quantity and/or binding affinity of the labeled first reagent can be made in order to determine the quantity of analyte present in the sample.
Sargeant, et al. (EP 0 296 724 A2) disclose an apparatus for determining the presence and/or amount of an analyte in a liquid sample. The patent discloses an apparatus comprised of a non-bibulous lateral flow membrane which is used as an assay substrate. The membrane has an application zone adapted to receive the sample. The membrane also includes an indicator zone spaced laterally from the application zone. The affixed end of the indicator zone is a member of a binding pair capable of binding the analyte or a derivative of the analyte. The patent teaches that the lateral spacing of the application zone and the indicator zone is configured such that liquid introduced at the application zone is caused to flow by non-bibulous flow through the indicator zone. Consequently, utilizing the device as disclosed in the patent, a sample is applied to the substrate and allowed to flow laterally through the indicator zone. Any analyte in the sample is complexed by the affixed specific binding member and detected. The patent discloses that by varying the amount of specific binding pair member in a multiplicity of indicator zones on the membrane, the assay can be made semiquantitative. Consequently, they teach that by calibrating serial dilutions of a binding pair member preparation, affixed over a series of indicator zones, to varying concentrations of analyte, at least a semiquantitative result can be obtained.
Gordon, et al. (EP 0 262 328 A2) disclose test strips for analysis of analytes such as antigens, antibodies, and polynucleotides in a sample by means of a sequential series of reactions. A strip is comprised of a length of chromatographic material having a first end, at which chromatographic solvent transport begins, and a second end, at which chromatographic solvent transport ends, and a plurality of zones positioned between the first and second ends. The first zone is impregnated with the reagent which is mobile in the solvent and capable of reaction with and immobilization against solvent transport by the analyte when the analyte is in an immobilized form. The second zone receives the sample. A third zone is impregnated with another reagent which is immobilized against solvent transport. The reagent is capable of selective reaction with the analyte so as to render the analyte in an immobilized form in the third zone. The patent generally discloses detection means wherein the presence of an analyte in a sample renders a positive color reaction and wherein the absence of analyte renders no color reaction.
Olson, et al. (EP 0 259 157 A1) describe a method for determining the presence of an analyte in a sample which includes utilizing a piece of bibulous material capable of being traversed in at least one direction by a test solution through capillary migration. The test solution is comprised of a sample, an antibody for the analyte, a conjugant of the analyte, and a label. The bibulous material contains a first receptor for the conjugant non-diffusively bound to a situs on the bibulous material separated from a contact portion. The contact portion of the bibulous material provides for contacting with the test solution. The bibulous material further contains a second receptor capable of binding the antibody for the analyte. The second receptor is non-diffusively bound to the bibulous material at least between the situs and the contact portion. The patent discloses that in practicing the method of the alleged invention, the contact portion of the bibulous material separated from the situs is contacted with the test solution which traverses the bibulous material by means of capillary action. After the test solution is allowed to traverse the bibulous material, the situs is examined for the presence of conjugate. The patent discloses that the situs can be exposed to a signal-producing means capable of interacting with the label to produce a signal in relation to the amount of analyte in the test solution.
Olson, et al. further disclose an embodiment wherein the first receptor is conjugated to particles which are non-diffusely bound to the bibulous material at the situs. The patent teaches that the situs can be a narrow or wide band running transverse to the direction of traversal of the test solution along the material. It is stated that a signal produced at the situs can be a narrow or wide band, a sharp-edged distinctive pattern, or the like. The signal generated at the situs can be compared with adjacent areas on the bibulous material. The patent teaches that in some quantitative assays, the signal can be measured directly at the situs without comparison to adjacent areas on the bibulous material.
Deutsch, et al. (EP 0 189 925 A2) describe a process for determining the presence of a particular immunological component in a test sample, comprising immobilizing at one end of a wick a specimen of said immunological component or of an immunological component specific to it. Passing the sample through the wick with a first reagent comprised of an enzyme conjugated to another immunological component which is either specific to the immunological test component or an immunologically comparable to the test sample, whereby the enzyme-carrying reagent attaches to antigen sites if such sites have not previously been filled by the immunological component in the test sample. The process disclosed includes then passing through the wick by capillary action a second reagent containing a substrate acted upon by the enzyme of the first reagent and determining the extent of enzymatic reaction which indicates the amount of immunological component in the initial sample. The extent of enzymatic reaction may be observed by a color change. Deutsch, et al. generally state that the color change or fluorescence may be qualitative, that is, yes or no as in a pregnancy test, or it can be quantitative.
Litman, et al. (EP 0 160 467 A2) describe a protocol which incorporates an enzyme substrate on a bibulous support to which is conjugated a member of a specific binding pair. Upon introduction of the support into the assay medium, the support diffuses into the medium, eliminating a separate development step for the assay. The patent discloses that the result of the assay is determined by a change in the development of color as a result of enzyme catalyzed product formation from the substrate.
Litman, et al. describe two devices: an immunochromatograph and a dip-stick. The dip-stick embodiment is stated to involve one or two elements, where one element is the sample element and the other element is the standard. The immunochromatograph involves a bibulous support providing liquid travel through capillarity, a non-diffusively bound MIP, and also includes one or more members of the signal producing system, i.e., at least one substrate. The immunochromatograph involves a bibulous support which provides a stationary solid phase and an assay medium which provides a moving liquid phase. The region in which the MIP is uniformly non-diffusively bound to the bibulous support is referred to as the immunosorbing zone. The analyte from the sample traverses the zone being carried along with a solvent whose front traverses the zone. The analyte, which is the homologous or reciprocal MIP to the MIP bound to the support, becomes bound to the support through the intermediacy of MIP complex formation. The signal producing system provides the manner in which the area in the immunosorbing zone to which the analyte is bound may be distinguished from the area in which it is absent, so that the distance from a predetermined point on the immunochromatograph is a measure of the amount of analyte in the sample.
Weng, et al. (U.S. Pat. No. 4,879,215) describe a method for determining the presence of an analyte in a sample suspected of containing the analyte wherein the analyte is a member of a specific binding pair consisting of a ligand and its complimentary receptor. The method includes contacting a bibulous material with a test solution containing the sample and a first specific binding pair member capable of binding to the analyte. The material contains a second specific binding pair member non-diffusively bound to a small situs on the material separated from the contact portion. The second specific binding pair member binds the first specific binding pair member when the first specific binding pair ("sbp") member is bound to the analyte. In addition, when the second sbp member is able to bind the first sbp member which is not bound to the analyte, an analyte analog capable of binding the first sbp member is non-diffusively bound to the material. The test solution is allowed to traverse the material by means of capillary migration and the presence of the first specific binding pair member is detected at the situs. The method includes a signal producing system which comprises a labeled-sbp member conjugate and other reagents required to produce a signal at the situs in relation to the presence or amount of analyte in the sample.
Bahar, et al. (U.S. Pat. No. 4,868,108) describe an assay employing two antibodies wherein a microporous carrier supports a plurality of layers, in which conjugates form between test antigens and antibodies. Quantitative results are said to be obtained from an immobilized antibody within an indicating substrate. An apparatus is described for indicating the test antigen in the solution comprised of the following: a porous support having a plurality of regions, a first antibody bound to an enzyme to form an enzyme-linked antibody disposed within one of the regions, a second antibody immobilized within a region different from the first antibody, and a substrate disposed in a region other than the regions of the antibodies which is capable of reacting with the enzyme to yield an indication of test antigen. In addition, the patent teaches that the regions are disposed such that solution flows through the region of the first antibody to the region of the second body and then to the region of the substrate.
Friesen, et al. (U.S. Pat. No. 4,861,711) describe a solid diagnostic device for the quantitative determination of substances of biological affinity in biological fluids. They describe a device comprised of a layer of a plurality of planar zones adjacent one another and in absorbent contact with one another. The layer includes application, intermediate, and absorption zones. The zones are positioned such that the liquid is capable of moving by absorption from the application zone through the intermediate zone onto the absorption zone. The intermediate zone comprises a solid phase zone having at least one unlabeled reactant capable of interactions of biological affinity with at least one analyte. Disposed in an area between the application zone and the solid phase zone is at least one unattached, labeled reactant or conjugant, capable of interactions of biological affinity with at least one analyte. In addition, the device includes an analyte application zone disposed at the mobile phase application zone or in between the mobile application zone and the absorption zone. The analyte application zone is disposed such that after application of an analyte, the analyte is reacted with the reactants in the layer and is detected in the layer.
Ullman, et al. (U.S. Pat. No. 4,857,453) describe a device for conducting an assay method. The device comprises a housing. Enclosed within the housing is a means for capturing a member of a specific binding pair in a zone and for allowing liquid to be transported by capillary action away from the zone. Also enclosed in the housing are one or more self-contained reagents for conducting an assay method for determining an analyte in a sample. In addition, the housing includes means for introducing a sample to the device. The patent teaches that preferably liquid reagents are combined in at least one breakable container enclosed in the housing. In relation to a quantitative readout, the patent teaches that the device may possess on its face a scale to assist in quantitating the amount of analyte in the sample.
Rosenstein, et al. (U.S. Pat. No. 4,855,240) describe a test device and assay for determining analyte. The device is constructed such that a tracer and a sample may be simultaneously applied to different absorbent material portions both in capillary flow communication with another absorbent material portion having a binder supported thereon. The device is constructed such that the sample contacts the binder prior to any substantial contact between sample and/or tracer and binder. Specifically, the patent discloses an assay employing an absorbent solid support having first, second, and third portions. The first portion receives a sample to be assayed. The second portion receives a tracer and said third portion includes a binder for the analyte. The third portion is in capillary flow communication with the first and third portions. The process includes moving tracer and sample by capillary flow across the binder and the third portion to provide contact between sample and binder prior to substantial contact of the tracer with either the binder or sample. The analyte is determined by determining at least one of the tracers bound in said third portion or tracer which passes across that third portion. The patent further discloses that the preferred tracer is comprised of a ligand and a particulate label, and the particulate label preferably provides a tracer which is visual under the assay conditions so that the presence and/or amount of analyte may be determined without further treatment and without the use of instrumentation, e.g., by use of a liposome containing a dye as the particulate label.
Wie, et al. (GB 2 180 645 A) describe a device to determine the presence of a minute amount of a specific substance in a liquid medium in the form of a test card which includes top and bottom sheets adhesively secured to an intermediate frame member which, together with the top and bottom sheets, defines a filter chamber housing a flat filter. The test portion of the filter includes a binding substrate to which antibodies of the specific substance have been bound. The patent further discloses that a control area on the filter body may be provided which is juxtaposed to the test area. The control area incorporates the same binding substrate and antibody as the test area and is located in the proximity to a control port. The control port is subjected to certain method steps of the invention to provide a control reading which can be compared with the test reading resulting from the administration of the test sample to the test port.
Perry, et al. (PCT/GB88/00329; WO 88/08536) describes an enzyme-labeled binding assay device comprised of an absorbent material and a developing solution containing a substrate for the enzyme. The material is provided with a plurality of reagent zones including an indicator reagent zone. The indicator reagent is capable of immobilizing an enzyme-labeled reagent in an amount dependent on the assay result. The absorbent material includes a reagent that prevents signal formation except where enzyme-labeled reagent is immobilized at the indicator reagent zone. The enzyme and substrate are chosen such that when in contact they produce a signal, such as color formation which may be readily determined. In addition, according to one embodiment described, the intensity of the signal at the indicator zone is inversely proportional to the concentration of the analyte in the sample. The device can be employed to provide quantitative assay results wherein the intensity of the signal produced at the indicator zone will be either proportional to or inversely proportional to the concentration of analyte present in the sample. Consequently, the indicator zone following an assay may be inserted into a reflectance spectrophotometer or a fluorometer, to measure the intensity of the signal produced.
Baker, et al. (PCT/GB 86/0067; WO 87/02774) describe a device for performing an enzyme-labeled binding assay comprised of an absorbent material and a developing solution. The absorbent material is provided with a plurality of reagent zones. One of the zones is an indicator reagent zone and is capable of transporting the developing solution sequentially through each reagent zone. The indicator reagent zone includes a reagent capable of immobilizing an enzyme-labeled reagent in an amount dependent upon the assay result. The developing solution includes a signal-producing substrate for the enzyme.
The results of an assay utilizing the disclosed device may be qualitative, read simply by the absence or presence of a signal, especially a colored signal at the indicator reagent zone. In addition, the device can be employed to provide quantitative assay results. The intensity of the signal-produced at the indicator reagent zone can be either proportional to or inversely proportional to the concentration of an analyte present in the sample. As a result, the indicator reagent zone of the device may, following an assay, may be inserted into a reflectance spectrophotometer, or a fluorometer to measure the intensity of the signal produced. In addition, the indicator reagent zone may be elongated in the direction of developing solution migration or a plurality of individual indicator reagent zones may be provided. Thus, the length of signal produced at the indicator reagent zone or the number of individual zones which exhibit this signal will be quantitative and proportional to, or inversely proportional to, the concentration of analyte in the sample.
Campbell, et al. (U.S. Pat. No. 4,703,017) describe a solid phase assay for an analyte wherein binder is supported on a solid support, such as nitrocellulose, and a tracer is comprised of a ligand labeled with a colored particulate label, such as a liposome including a dye. The assay described in this patent has a high sensitivity, and the tracer is visible on the support under assay conditions, whereby tracer can be determined without instrumentation, and without further treatment thereof.
In FIG. 1, the Campbell, et al. patent discloses a test substrate, in the form of a sheet, which is formed preferably from nitrocellulose. The sheet includes three specified distinct test areas each of which is provided with binder. The binder may be an antibody, and the analyte can be a hapten or antigen. The antibody is applied to the three test areas. The remainder of the test strip or sheet includes a suitable blocking agent to prevent non-specific binding. The patent discloses that the three test areas may be provided with antibodies to the analyte having a different affinity for the analyte, with one test area having a high affinity for the analyte, another test area having a moderate affinity for the analyte, and the third test area having a low affinity for the analyte. Alternatively, the test areas may be provided with antibody having the same affinity for the analyte, with the first test area having a high concentration of antibody, the second test area having a medium concentration of antibody, and the third test area having a low concentration of antibody.
Rosenstein (EP 0 284 232 B1) describes an assay device comprising a test strip having a first portion and a second portion. The first and second portions are in the same plane such that material can flow by capillary action from one portion to the other. The first portion includes a tracer comprised of a ligand (specific for the analyte when the device is configured for a sandwich assay and is the analyte or analog thereof when the device is configured for a competitive assay) conjugated to a non-soluble particulate marker. The first portion is also the situs for the addition of the sample to the device. The second portion has a binder immobilized in it (which is specific for the analyte when the device is configured for a sandwich assay and is specific for the analyte and ligand when the device is configured for a competitive assay). The binder is present in the second portion in an amount sufficient such that the tracer bound in that portion is visible. This patent teaches that in an assay, the determination of a tracer and/or analyte may be either qualitative or quantitative.
Donovan, et al. (PCT/US93/08417; WO 94/07141) describe assay devices and methods utilizing dual pathways which provide for the confirmation of assay results. According to one embodiment disclosed, a sandwich-type assay device is described which includes a solid phase having at least two separate fluid-flow pathways. The first pathway contains an assay capture site and the second pathway contains both a confirmatory reagent and a confirming assay site. The first and second pathways may be two individual pieces of porous material or two separate portions of a single porous material. A specific binding member which binds a labeled analyte complex is immobilized both in the first pathway at the assay capture site and in the second pathway at the confirming assay site. The device further contains a confirmatory reagent involving an unlabeled specific binding member which binds the analyte and inhibits the binding of the analyte or labeled analyte complex in the confirming assay site. After the test sample is contacted to the device, the labeled analyte complex becomes immobilized within the first pathway at the assay capture site to indicate the presence or amount of an analyte in the test sample. In the second pathway, the confirmatory reagent blocks the binding of the analyte or labeled analyte complex to the confirming assay site, thereby confirming that the presence of the label in the assay capture site indicates a positive test sample.
Also disclosed is a competitive assay device which includes a solid phase having at least first and a second separate fluid-flow pathways. The first pathway contains an assay capture site and the second pathway contains both a confirmatory reagent and a confirming assay site. An immobilized specific binding member, which competes with the analyte for binding a labeled reagent, results in the binding of the labeled reagent in inverse proportion to the presence or amount of the analyte in the test sample. The binding member is immobilized both in the first pathway at the assay capture site and in the second pathway at the confirming assay site. The device further contains a confirmatory reagent comprising an unlabeled specific binding member which blocks the binding of the analyte in the confirming assay site. The test sample is contacted to the device, and a confirmed positive result is one in which the device displays a decrease in signal or no signal at the assay capture site whereas the confirming assay site displays a detectable signal. The patent teaches that in a preferred embodiment, a visually detectable label is used as the label component of the labeled reagent, thereby providing for the direct visual or instrumented readout of the presence or amount of analyte in the test sample without the need for additional signal producing components at the detection sites.
Pronovost, et al. (EP 0 613 005 A2) describe test articles which comprise a support matrix, and a labeling complex impregnated within the support matrix, wherein the labeling complex comprises a dyed microorganism having a specific binding substance on its cell surface. The patent teaches the accumulation of visible microorganism label may be assessed either visually or by optical detection devices, such as reflectance analyzers and video image analysis and the like. The accumulation of visible label can be assessed either to determine the presence or absence of a label in the capture zone or the visible intensity of accumulated label may be correlated with the concentration of analyte in the biological sample. The correlation between the visible intensity of accumulated label and analyte concentration may be made by comparison of the visible intensity to a reference standard which may or may not be incorporated into the capture zone. Optical detection devices may be programmed to automatically perform this comparison. Visual comparison is said to also be possible by visual evaluation of the intensity with a color key. Swanson, et al. (U.S. Pat. No. 4,073,484) describe a method and apparatus for the quantitative determination of an analyte in a liquid sample. The method and apparatus employ a liquid-permeable solid medium defining a liquid flow path. The medium includes a number of reagent-containing reaction zones spaced apart along the flow path and in which reaction occurs with the analyte or an analyte derivative to result in the formation of a predetermined product. Detector means are employed to detect analyte, analyte derivatives, reactant or predetermined product in the reaction zones, the number of such zones in which such detection occurs indicates the amount of analyte in the liquid. Thus, there is a need for an assay which permits the easy simultaneous qualitative and quantitative determination, e.g., without specific detection and reaction zones, of an analyte by the use of a single device wherein the determination can be made visually without instrumentation and without further treatment.